POS1332 ANTI-NUCLEAR VALOSIN-CONTAINING PROTEIN-LIKE AUTOANTIBODIES ARE ASSOCIATED WITH A NEW CLINICAL PHENOTYPE IN SYSTEMIC SCLEROSIS PATIENTS: LIMITED CUTANEOUS SUBSET WITH RECALCITRANT CALCINOSIS AND HIGHER RISK OF CANCER
Authors: J. Perurena-Prieto, L. Viñas-Giménez, M. T. Sanz-Martínez, A. Selva-O’callaghan, E. L. Callejas-Moraga, R. Colobran, A. Guillen-Del-Castillo, C. P. Simeon Aznar
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> It has been reported that 80-85% of established systemic sclerosis (SSc) patients present anti-nuclear autoantibodies (ANAs) [1]. However, in a group of clinically heterogeneous patients with high-titer ANAs no SSc-specific autoantibodies are found. It is likely that these patients present autoantibodies that have not been previously described. Protein immunoprecipitation (IP) followed by mass spectrometry (MS) has the potential to identify these new autoantibodies. <h3>Objectives</h3> To identify novel autoantibodies through a new protein IP assay based on bioorthogonal non-canonical amino acid tagging (BONCAT) combined with MS and to describe the clinical associations of these novel autoantibodies. <h3>Methods</h3> Sera samples and clinical data of 307 SSc patients from Vall d'Hebron University Hospital were collected. All patients met the LeRoy and Medsger criteria and 84.0% fulfilled the 2013 ACR/EULAR classification criteria of SSc. ANAs were tested in all patients by indirect immunofluorescence (IIF) on HEp-2 cells and positive reactions were categorized according to the International Consensus on ANA Patterns (ICAP) classification. Specific autoantibodies (anti-Scl70, -centromere, -RNA-polymerase III, -fibrillarin, -NOR-90, -Th/To, -PM/Scl, -Ku, -U1-RNP, -U11/U12-RNP) were evaluated by a commercial immunoblot and chemiluminescence immunoassay and traditional RNA IP. Patients negative for all tested autoantibodies (n=51) were further studied by an IP assay based on BONCAT. Protein IP was performed by incubation of sera of these patients with protein A-sepharose beads in IP buffer, and subsequent incubation with labelled K562 cell extracts. Obtained immunoprecipitates were fractionated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands detected on SDS-PAGE were further analysed by Liquid Chromatography-MS. <h3>Results</h3> Five patients tested by protein IP showed a band with a molecular weight of 110-115kDa on SDS-PAGE and a homogeneous nucleolar pattern (AC-8) by IIF. These bands were identified as nuclear valosin-containing protein-like (NVL) by MS. All anti-NVL positive patients fulfilled the 2013 ACR/EULAR classification criteria of SSc and presented the limited cutaneous (lcSSc) subtype. Anti-NVL positive patients were characterised by higher age at SSc onset (51.7 vs. 39.9 years, p=0.041) and higher prevalence of calcinosis (100% vs 10.9%, p&lt;0.001). More importantly, anti-NVL positive patients presented higher risk for malignancy (60.0% vs 4.3%, p=0.005; OR 33.0; 95% CI 3.4-323.4) and synchronous cancer (40.0% vs 2.2%, p=0.023; OR 30.0; 95% CI 2.1-433.1). <h3>Conclusion</h3> We have identified NVL as a novel autoantibody target on SSc patients by a new protein IP assay. Anti-NVL positive patients presented a lcSSc phenotype characterized by high prevalence of calcinosis and higher risk of cancer, including synchronous cancer. Further work is required to confirm the clinical associations that were found in this cohort of SSc patients and to evaluate the presence of anti-NVL in other autoimmune diseases and conditions. <h3>Reference</h3> [1]Denton CP, Khanna D. Systemic sclerosis. Lancet. 2017;390(10103):1685–99. <h3>Acknowledgements</h3> The authors are grateful to Professor Ricardo Pujol-Borrell (Vall d'Hebron University Hospital), for his collaboration in the development of this project. Dr. Vicent Fonollosa (Vall d'Hebron University Hospital), for his commitment to the establishment of the systemic sclerosis research line at our hospital. We also thank the team of Professor Neil McHugh (University of Bath) for sharing their knowledge about protein immunoprecipitation with us. <h3>Disclosure of Interests</h3> None Declared.